1. Field of the Invention
This invention discloses a novel method for the in vitro culture and proliferation of Insecta germ cells by the application of soluble cytokinic and mitogenic agents.
2. Background Art
All insect cells were once thought to be impossible to culture, fully half a century passed between the first successful mammal cell cultures and 1962 when Grace finally formulated a medium capable of supporting insect cells in vitro. Even today investigations of insects cells lags far behind. Mice, humans, pigs, and birds receive the vast share of research attention. Only with the baculovirus expression system have a select number of insect tissue cells found a significant role as minute disposable factories for producing invaluable medical and other proteins. Large numbers of these tissue cells have been established in collections to support this particular system in its various forms. Notably absent from these tissues collections are any insect cells cultures or lines derived from germ cells. All germ cell cultures have proven very difficult to culture and successfully propagate; only murine (mouse). avian (chicken) and porcine (pig) embryos have yielded sustained germ cell cultures that have been reported in the literature. (Margolis J. and A. Spradling, 1995. Development 121:3797-3807: Potten, C. and M. Loeffler, 1990. Development 110:1001-1020).
In contrast to the complete lack of success in culturing insect germ cells, the 1980's first saw murine germ cells cultured with limited success on mouse fibroblast and buffalo rat liver cells. This early work was soon followed in the early 1990's by complete success reported by several teams in diverse locations within a matter of months of each other. Petitte was the first to achieve an avian (chicken) germ cell culture in the same period. Advances in murine and avian germ cell culture were made possible by a new array of compounds that demonstrated enhanced survival and growth characteristics when applied to primordial germ cells. Dolci, Matsui, Godin, Resnick and Hogan all reported various results culturing murine germ cells with several mediums and feeder layers supplemented with diverse materials typically including stem cell factor, leukemia inhibitory factor, and fibroblast factors in combination with numerous other agents and techniques. (Evans, M. et al., 1981. Nature 292:154: Smith, A. et al., 1987, Developmental Biology 121:1-9: Petitte, U.S. Pat. No. 5,340,740(1994): Petitte, U.S. Pat. No. 5,656,479(1997): Dolci, S. et al., 1991, Nature 352:809-811: Dolci, S. etal., 1993, Molecular Repro. Dev. 35: 134-139: Matstui, Y. etal., 1991. Nature 353:750-752: Godin. I. et al., 1991. Nature 352:807-809: Williams, U.S. Pat. No. 5,166,065 (1992Resnick. J. et al., 1992. Nature 359:550-551: Hogan, U.S. Pat. No. 5,453,357 (1995). Progress slowed following this burst of achievement, little use and no advancement of germ cell culturing methods has been reported for murine and avian cultures since. After a lapse of six more years Shim and Piedrahita reported independently in late 1997 and early 1998 that porcine embryonic germ cells had been successfully cultured. (Shim. H. et al., 1997. Biology of Reproduction 57 (5): 1089-1095: Piedrahita. J. et al., 1998, Biology of Reproduction 58 (5): 1321-1329).
While success has been reported for murine, avian and porcine germ cell cultures there are no reports of success establishing a method for insect germ cells culturing: the pattern of slow progress for insect cell research established in the first half of the century is repeating itself. There are reasons for this delay. Insect cells are distinctly different from other animal cell and comparatively little is known about insect cell metabolism and beyond observational data equally little is known about influencing or culturing their germ cells. There is a relatively small body of literature on the subject. Insect and vertebrate animals are classified in totally different phylogenetic systems. Insect cells developed in a substantially older and distinctly more primitive evolutionary period than that experienced much later by avians and most recently by modem mammals. Insects have a radically dissimilar and unique life cycle resulting from distinct cellular properties. Basically, insect cells often react in different and unanticipated ways when techniques and materials developed for a few select vertebrates are applied to them.
The best known example of how insect cells are fundamentally different from mammal cells is the basis of the baculovirus expression system. Early investigators tried repeatedly to extract recombinant intact human plasminogen from a number of different cell types and each attempt failed. (Whitefleet-Smith, J. et al., 1989, Arch. Biochem. Biophs. 271:390-399). One known unique cellular property inherent to insect cells is the lack of intracellular plasminogen activators, a feature found in mammals. Mammal cells with activators rapidly convert plasminogen to plasmin before the material can be recovered and consequently make an expression system based on mammal cells essentially useless. Insect cells, lacking these activators, can create and maintain significant amounts of recombinant human plasminogen with relative efficiency before lysis. Insect cells have many radically distinct characteristics, known and mostly unknown, which absolutely preclude any confident assumption about transferring knowledge learned from manuals to conclusions concerning insects.
The culture requirements of insect cells further illustrate these differences. In culture insect cells have a greater oxygen uptake rate than mammalian cells. The insect cell's membranes are comparatively fragile. surfactants are needed to protect and strength the membranes against the minute hydrodynamic forces of most cultivating systems. Insect cells grow optimally at about 27.degree. C., mammals prefer closer to 37.degree. C. Media for culturing insect cells needs to be set at a 6.2 pH, mammalian cell require a 7.4 pH. Most insect germ cells never mature but instead become a nutritive attachment to those cells that do become oogonia; there is simply no equivalent system in the formation of mammalian or avian germ cells. These few illustrative facts indicate the scope of the dissimilarities between the cells of most animal insect cells, and specifically insect germ cells.